Metagenomic Analysis of Cystic Fibrosis Sputum
Cystic fibrosis (CF) is an autosomal recessive genetic disorder that affects lung, pancreas, liver and intestine 4. In European Union, one out of 2000-3000 newborns is found to be affected by CF 2. CF is caused by mutations of the gene encoding cystic fibrosis transmembrane conductance regulator (CFTR), a cyclic AMP-dependent channel protein controlling the transport of chloride and sodium ions across the epithelial membrane 4. There are over 1500 possible mutations associated with CF. ΔF508, a deletion mutation of a phenylalanine at 508th position of CFTR is the most common mutation and accounts for about two-thirds of all CF cases 2. This deletion mutation causes the malfunctioning and loss-of-function of CFTR, thus the chloride ion and water cannot flow out from the cytoplasm, leading to the thick mucus in the lower respiratory tract 3. The accumulation of viscous secretion will lead to the blockage of the narrow passage and the subsequent remodeling of the affected lungs. Meanwhile, these blockages form a suitable reservoir for the colonization and reproduction of opportunistic pathogens . There are two functional microbial communities in CF lungs: Climax ''and ''Attack ''communities 1. ''Attack ''community refers to the microbes newly acquired that induce strong innate immune response and trigger acute pulmonary exacerbations. ''Climax community is the microbes that are stable over treatment and often resistant to therapies. Metagenomics is the study of genomic of a complex sample. 454 sequencing is a large-scale parallel pyrosequecing normally used in megtagenomic analysis. A metagenomic study was performed to analyze the sputum samples from a CF patient in four different time points and to study the change of the microbe community in the sputum sample 1. Sample collection and workflow: Before sample collection, the patient rainsed her mouth with sterile saline to minimize the contamination of oral microbes. The sputum sample was collected after the inhalation of 4 ml 7% hypertonic saline via a nebulizer 1. The sample was then treated with β-mercaptoethanol to homogenize the mucus. The sample was washed with distilled water to lyze human cells and then treated with DNase to degrade the extracelluar DNA from lysed human cells and the extracellular matrix of the biofilm. The total DNA was extracted using a Gram-positive bacteria genomic DNA extraction kit and sequenced by 454 sequencing 1. Results: The four samples collected are: A: before the administration of antibiotics; B: before the change of antibiotics; C: when the symptoms resolved and D: when the patient was clinically stable 1. Changes of microbe community in the sputum sample: Escherichia coli is the major microbe in the first sample analyzed. Two microbes that are not commonly associated with CF, Streptococcus parasanguinis and Rothia mucilaginosa, are found existing in the sample 1. With the antibiotic treatment going on, the total number of microbes decreased dramatically. The relative abundance of E. coli ''decreased during the treatment and slightly increased after the treatment. The ''Strpetococcus spp. population significantly increased during the treatment but decreased after the treatment. The relative abundance of R. mucilaginosa increased during the treatment. After the antibiotic treatment, E. coli ''and ''R. mucilaginosa become dominant in the sample 1. Changes of antibiotic resistance in the sample: ] The patient was treated with two sets of antibiotics including beta-lactams, macrolides, aminoglycosides and so on 1. Thus the microbial community developed antibiotic resistance during the treatment. Metagenomic data suggested the existence of multiple antibiotic resistance strategies: multidrug resistance efflux pumps and a variety of enzymes that can give antibiotic resistance (e.g. β-lactamase). Increased abundance of gene'' aac'' and blaA are also found from the metagenomic data, indicating the'' Climax'' microbes gained more antibiotic resistence compared to the attack microbes 1. References： 1. Lim, Y. W. et al. Clinical Insights from Metagenomic Analysis of Cystic Fibrosis Sputum. J. Clin. Microbiol. (2013). doi:10.1128/JCM.02204-13 2. Folkesson, A. et al. [http://www.nature.com/nrmicro/journal/v10/n12/full/nrmicro2907.html Adaptation of Pseudomonas aeruginosa to the cystic fibrosis airway: an evolutionary perspective. ] Nat. Rev. Microbiol. 10, 841–51 (2012). 3. Murray, T. S., Egan, M. & Kazmierczak, B. I. ''Pseudomonas aeruginosa ''chronic colonization in cystic fibrosis patients . (2007). 4. http://en.wikipedia.org/wiki/Cystic_fibrosis